Project Summary Melanoma is the most aggressive form of skin cancer and, if disseminated through the dermis, has a poor prognosis with a high mortality rate. Hypoxia is a component of the tumor microenvironment, which reduces efficacy of both immuno- and chemo-therapies resulting in poor clinical outcome. Therefore, we will exploit the hypoxic microenvironment as a target for gene therapy, utilizing commensal facultative anaerobic bacteria. The overall goal of this proposal is to develop an effective and safe delivery system for cancer gene therapy by targeting the hypoxic tumor microenvironment with food-grade lactic acid bacteria (LAB) Lactococcus lactis (L. lactis). The use of LAB, as opposed to other bacteria, represents a much more desirable strategy to deliver therapeutic genes than attenuated pathogenic strains, as there is no risk of reversion or potential to instigate host reactions in immunocompromised patients. Because identification of tumor-specific accumulation and biodistribution of gene delivery vehicles in vivo is essential for translation of these agents to the clinic, we will utilize a newly emerging technology, multispectral optoacoustic tomography (MSOT). MSOT is a hybrid modality that detects sound waves generated by the absorption of electromagnetic energy, thus enabling the capability for 3D high-resolution at depth and in real time. Our preliminary data indicates that L. lactis expresses high levels of -galactosidase (LacZ), and in combination with 5-bromo-4-chloro-3-indolyl-?-D- galactopyranoside (X-gal), produces a strong blue color to facilitate detection of L. lactis using MSOT. Given these findings, we hypothesize that L. lactis will preferentially colonize the hypoxic areas of the tumor microenvironment to deliver therapeutic genes in a safe, tumor-specific, and effective manner. We propose two aims: (1) Evaluate the efficacy of L. lactis as a gene delivery vehicle in melanoma cells in vitro, and (2) determine the biodistribution and tumor-specific accumulation of L. lactis in metastatic melanoma-bearing mice. The successful completion of this proposal will substantially influence the field of gene therapy, specifically utilization of facultative bacteria as delivery agents for tumor-specific targeting of melanoma.